All of the cells and cell types of an individual adult mammal are derived from a single cell, the zygote. However, as cells mature and differentiate they lose their ability to be converted into a different cell type. Thus, most adult cells are fully differentiated and normally cannot be converted into another cell type. One particular exception is the adult stem cell. Adult stem cells retain the ability to differentiate into other cell types, though this differentiation is generally limited to forming cells of a single tissue type. For example, hematopoietic stem cells (HSCs) are capable of differentiating into any cell type of the blood and immune system, whereas brain stem cells can differentiate into the different cell types of the brain. In recent years, therapies for treating degenerative diseases and/or cancer (such as leukemia) have been designed which employ stem cells. However, heretofore, isolating stem cells from the human donors has proved to be extremely difficult since stem cells are relatively rare.
Hematopoietic stem cells are functionally defined based on their capacity for self-renewal divisions, which leads to the continuous generation of new HSCs over the lifetime of an animal, and by their potential for pluripotent hematopoietic differentiation. There are three possible general outcomes for the resulting daughter cells when a hematopoietic stem cell divides: (i) differentiation, (ii) self-renewal, or (iii) apoptosis. Despite the extensive study of HSCs, due to its relevance to bone marrow transplantation, gene therapy, and basic hematopoiesis, the mechanisms controlling these three tightly regulated outcomes are poorly understood.
Purification strategies for HSCs have been developed for both mouse [Spangrude et al., Science 241:58-62 (1988):(published erratum appears in Science 244(4908):1030 (1989)); Uchida et al., J.Exp.Med. 175:175-184 (1992)] and humans HSCs [Zanjani et al., J. Clin.Invest. 93:1051-1055 (1994), see comments; Larochelle et al., Nat.Med. 2:1329-1337 (1996); Civin et al., Blood 88:4102-4109 (1996)]. Most of these strategies use antibodies directed against various cell surface antigens and multiparameter cell sorting to isolate phenotypically defined cell populations. This approach has allowed isolation of murine stem cell populations of sufficiently high purity to allow reconstitution of irradiated recipients with less than 10 cells [Morrison et al., Proc.Natl.Acad.Sci. USA 92:10302-10306 (1995); Osawa et al., Science 273:242-245 (1996)], while considerably greater numbers of sorted human cells have been required to reconstitute xenogeneic recipients [Larochelle et al., Nat.Med. 2:1329-1337 (1996); Zanjani et al., Exp.Hematol. 26:353-360 (1998), see comments.]
The human MDR1 gene and its murine homologs were originally identified based on the ability of their expressed products, collectively referred to as P-glycoproteins (P-gps), to extrude a wide variety of cytotoxic drugs from the cell interior [Gros et al., Cell, 47:371-380 (1986) and Chen et al., Cell, 47:381-389 (1986)]. It is now known that the MDR1 gene belongs to a superfamily of transport proteins that contain a conserved ATP-binding cassette (ABC) which is necessary for pump function [Allikmets et al., Hum. Mol. Genet. 5:1649-1655 (1996)]. Numerous studies have clearly shown that P-gp expression plays an important role in the resistance of human tumor cells to cancer chemotherapy [Pastan and Gottesman, Annu. Rev. Med., 42:277-286 (1991)]. Considering that P-gps are also expressed in a wide variety of normal tissues, more recent studies have examined the normal physiologic functions of MDR1-like genes. Murine gene disruption experiments have demonstrated that expression of various P-gps is necessary for biliary excretion [Smit et al., Cell, 75:451-462 (1993)], maintenance of the blood-brain barrier [Schinkel et al., Cell, 77:491-502 (1994)], and elimination of drugs [Schinkel et al., Proc.Natl.Acad.Sci. USA, 94:4028-4033 (1997)]. P-gps can also mediate more general cellular functions including the translocation of lipids across the cell membrane [van Helvoort et al., Cell, 87:507-517 (1996)] and modulation of specific apoptosis pathways [Johnstone et al., Blood, 93:1075-1085 (1999) and Smyth et al., Proc.Natl.Acad.Sci. USA, 95:7024-7029 (1998)].
P-gp is expressed in a variety of hematopoietic cell types [Drach et al., Blood, 80:2729-2734 (1992)], including human CD34+stem cells [Chaudhary and Roninson, Cell, 66:85-94 (1991)] and murine c-kit+ stem cells [Sorrentino et al., Blood, 86:491-501 (1995)]. Several lines of evidence suggest that P-gp expression is functionally conserved in hematopoietic stem cells.
Another ATP transport protein that contains a conserved ATP-binding cassette is the gene product of the Bcrp1/Mxr/Abcp/ABCG2 gene (referred to herein as BCRP when obtained from any mammalian source, but as mBCRP and huBCRP when the specific mouse or human gene or gene product(s) are being particularly referenced). The huBCRP cDNA was originally cloned from several different human tumor cell lines that were resistant to multiple drugs including doxorubicin, topotecan, and mitoxantrone [Doyle et al., Proc.Natl.Acad.Sci. USA 95:15665-15670 (1998):(published erratum appears in Proc Natl Acad Sci U S A; 96(5):2569 (1999)); Maliepaard et al., Cancer Res. 59:4559-4563 (1999); Miyake et al., Cancer Res. 59:8-13 (1999)]. A highly related mouse homologue (mBcrp1) was cloned from fibroblasts selected for multidrug resistance [Allen et al., Cancer Res. 59:4237-4241 (1999)]. In contrast to the structure of the MDR1 gene, which consists of two duplicated halves, the predicted structure of BCRP is that of a “half transporter”, with a single ATP binding cassette and transmembrane region. The expression pattern of human BCRP (huBCRP) is highly restricted in normal human tissues, with the highest levels of mRNA detected in the placenta, and much lower levels detected in adult organs [Doyle et al., Proc.Natl.Acad.Sci. USA 95:15665-15670 (1998):(published erratum appears in Proc.Natl.Acad.SciU S A. 96(5):2569 (1999)); Allikmets et al., CancerRes. 58:5337-5339 (1998)].
Hematopoietic stem cells can be identified based on their ability to efflux fluorescent dyes that are substrates for P-gp, such as Rhodamine (Rho) 123 [Spangrude and Johnson, Proc.Natl.Acad.Sci. SA, 87:7433-7437 (1990); Fleming et al., J. Cell Biol., 122:897-902 (1993); Orlic et al., Blood, 82:762-770 (1993); and Zijlmans et al., Proc.Natl.Acad.Sci. USA, 92:8901-8905 (1995)] and Hoechst 33342 [McAlister et al., Blood, 75:1240-1246 (1990); Wolf et al., Exp. Hematol., 21:614-622 (1993); and Leemhuis et al., Exp. Hematol., 24:1215-1224 (1996)]. One particular approach for purifying stem cells is based on Hoechst dye-staining of bone marrow cells to identify a minor fraction of side population (SP) cells that are highly enriched for repopulating activity [Goodell et al., J. Exp. Med., 183:1797-1806 (1996)]. This SP phenotype identifies a primitive subset of stem cells present in multiple mammalian species [Goodell et al., Nat. Med., 3:1337-1345 (1997)], and based on verapamil inhibition studies, may be due to expression of P-gp or another ABC transporter [Goodell et al., J. Exp. Med., 183:1797-1806 (1996)].
Despite a recent report demonstrating that sorting for expression of the vascular endothelial growth factor receptor can enrich human stem cells to near purity [Ziegler et al., Science 285:1553-1558 (1999)], there still remains a general need for better and more specific markers of human HSCs. In addition, there is a great need for new methodologies of isolating stem cells.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.